Neuroblastoma is a neoplasm known to show spontaneous histologic maturation in vivo which correlates with a better biologic behavior and prognosis. Extracellular matrix (ECM) proteins, on the other hand, have been shown to influence tumor invasion and metastasis. Production of EMC proteins has been previously reported only for the C1300 murine neuroblastoma cell line. We have studied ECM synthesis (ie, fibronectin (FN), laminin (LM), and collagen type IV) in relation to differences in ECM protein synthesis by neuroblastomas in vitro before and after differentiation have been assessed by immunofluorescence, polyacrylamide gel electrophoresis and quantitative scanning densitometry of autoradiograms of these gels. Differentiation has been induced by dibutyryl-cyclic AMP and retinoic acid and studied by light and electron microscopy as well as biochemical expression of neurotransmitter enzymes. Finally, the biologic behavior of the neuroblastoma cells before and after differentiation with the above agents was tested in vitro, employing a human amnion invasion assay. Our results indicate that neuroblastoma shows tripartite differentiation in vitro into three main cell types: neuronal, Schwannian and melanocytic. Each cell type has different light and electron microscopic characteristics and exhibits a specifc pattern in terms of EMC protein expression. Quantitative studies of the synthesized EMC proteins showed definite changes of all three studied proteins with differentiation. Qualitative and quantitative changes of the neurotransmitter enzymes were also noted. Differentiation was correlated with decreased invasiveness in vitro.